Thermo Fisher Scientific QuantiGene User Manual

QuantiGene™ Plex Gene Expression Assay
USER GUIDE
Publication Number MAN0017862
Revision C.0
For Research Use Only. Not for use in diagnostic procedures.
Bender MedSystems GmbH | Campus Vienna Biocenter 2 | 1030 Vienna, Austria For descriptions of symbols on product labels or product documents, go to thermofisher.com/symbols-definition.
The information in this guide is subject to change without notice.
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Revision Date Description
C.0 02 March 2021 Updated the reagent volumes in the tables.
B.0 20 February 2020 Updated manufacturing address
A.0 27 August 2018 New document
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©2021 Thermo Fisher Scientific Inc. All rights reserved.

Contents

Product information ................................................................... 5
Product description ............................................................. 5
How the QuantiGene™ Plex Assay works ...................................... 5
Product Description ......................................................... 5
Precautions and technical hints ............................................... 6
Required equipment and materials not provided ................................ 6
Contents and storage ............................................................ 7
QuantiGene™ Plex Assay kit .................................................. 7
QuantiGene™ Plex Panel ..................................................... 8
Before you begin ...................................................................... 9
Before first use ................................................................. 9
Sample preparation ............................................................. 9
Assay procedure: day 1 ............................................................. 10
For cell lysates or whole blood lysates ............................................ 10
For fresh, frozen, or FFPE tissue homogenates ..................................... 12
For purified RNA or in vitro transcribed RNA ....................................... 14
Assay procedure: day 2 ............................................................. 16
Setup of the Luminex™ protocol .................................................. 16
Process Plate .................................................................. 16
Analyze results ....................................................................... 20
APPENDIX A Troubleshooting .................................................... 22
APPENDIX B Magnetic plate washer validation protocol ...................... 24
Validate the handheld magnetic plate washer ...................................... 24
QuantiGene
Plex Gene Expression Assay User Guide
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Contents
Sample optimization protocol ...................................................... 25
Optimize sample input .......................................................... 25
Determine the optimal lysis method for a sample type .............................. 26
APPENDIX C Safety ............................................................... 27
Chemical safety ................................................................ 28
Biological hazard safety ......................................................... 29
Documentation and support ....................................................... 30
Customer and technical support ................................................. 30
Limited product warranty ........................................................ 30
Glossary .................................................................................. 31
4
QuantiGene™ Plex Gene Expression Assay User Guide
IMPORTANT! Before using this product, read and understand the information in the “Safety” appendix
in this document.

Product description

How the QuantiGene™ Plex Assay works

The Invitrogen™ QuantiGene™ Plex Assay Kit enables the multiplexed measurement of gene expression by combining branched DNA (bDNA) signal amplification with Luminex™xMAP™ multi-analyte profiling technology. The bDNA assay is a probe hybridization-based method of target-specific RNA capture and quantitation, amplifying signal rather than the target.
Color-coded fluorescent magnetic microspheres (capture beads) capture specific target RNA molecules through hybridization of a custom oligonucleotide probe set, which consists of 3 types of probes: capture extenders, label extenders, and blocking probes. The probe set hybridizes a contiguous sequence of each target RNA. The capture extenders discriminate between dierent capture beads based on the sequence of a capture probe conjugated to each bead, and the label extenders have tails that provide the support for the branched DNA signal amplification.

Product information

Each amplification unit is constructed through sequential hybridization of bDNA oligonucleotides (pre­amplifier, amplifier, and label probe). The label probe is biotinylated to bind Streptavidin-conjugated R-Phycoerythrin (SAPE). The resulting fluorescence signal is associated with individual capture beads by the Luminex™ instrument, which combines advanced fluidics, optics, and digital signal processing. Signal is reported as median fluorescence intensity (MFI) and is proportional to the number of target RNA molecules present in the sample.

Product Description

The QuantiGene™ Plex Assay Kit consists of 3 modules, each sold separately:
QuantiGene™ Sample Processing Kit: contains reagents for release and stabilization of sample RNA from cultured cells, blood (whole blood, PAXgene™ blood, Tempus™ blood, or dried blood spots), or tissues (fresh, frozen or FFPE). These kits are not required if working with purified RNA samples
QuantiGene™ Plex Assay Kit: contains the generic reagents, plates, and seals required for running the assay
QuantiGene™ Plex Panel: contains the custom target-specific pooled probe set and associated magnetic capture beads to capture user-defined genes of interest
QuantiGene
Plex Gene Expression Assay User Guide
5
Product information
Product description
This user guide contains instructions for using the QuantiGene™ Plex Assay with the following sample types:
Cell lysates from cultured cells and whole blood
Tissue homogenates from fresh, frozen, or formalin-fixed, paran-embedded (FFPE) tissues
Purified or in vitro transcribed (IVT) RNA
For instructions on preparing cell lysates or tissue homogenates, please refer to the appropriate QuantiGene™ Sample Processing Kit package insert.

Precautions and technical hints

The shaking incubator must be calibrated for both 54°C & 50°C using the Temperature Validation Kit. The Vortemp 56 requires an inverted plate lid to be placed below the assay plates. See instructions for temperature calibration in the Temperature Validation Kit package insert.
When running a new sample type, optimize input by running a dilution series to ensure that all target signals are within the dynamic range of the assay.
Run samples in technical replicates. We recommend a minimum of duplicates, but ideally more in order to calculate intra-assay precision.
Use fresh pipette tips when loading samples into each well. Avoid creating bubbles when pipetting. Use a multi-channel pipette whenever possible to achieve optimal assay precision.
Be careful not to invert the plate or allow contents from one well to mix to another well. The Magnetic Separation Plate is to be inverted only when removing reagents and wash buer with the Handheld Magnetic Plate Washer.
On day 2 of the assay, turn on and initiate startup protocol of the Luminex™ instrument according to the manufacturer's instructions. Lasers require 30 minutes to warm-up.

Required equipment and materials not provided

Unless otherwise indicated, all materials are available through thermofisher.com. "MLS" indicates that the material is available from fisherscientific.com or another major laboratory supplier.
Required Equipment/Material
Handheld Magnetic Plate Washer EPX-55555-000
Microtiter plate shaker (must have 3 mm orbit at 600-800 rpm)
Vortex mixer MLS
Adjustable single and multi-channel precision pipettes for dispensing 1-20 uL, 20-200 uL, and 200-1000uL
Reagent reservoirs (25 mL and 100 mL capacity) 3054-1002 or equivalent (VistaLab
Nuclease Free Water (H2 O) MLS
Plate centrifuge capable of 240 × g speeds MLS
88880023 or 88880024, QP0706 (IKA™MS3
CLS4873 or equivalent (Corning™Costar™)
Source
Digital)
MLS
Technologies™)
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QuantiGene™ Plex Gene Expression Assay User Guide
Product information

Contents and storage

(continued)
Required Equipment/Material Source
Microcentrifuge MLS
QuantiGene™ Incubator Temperature Validation Kit QS0517
4" Soft Rubber Roller QS0515
Use one of the following:
FLEXMAP 3D™instrument APX1342
Luminex Corporation (sold through Thermo
Fisher Scientific)
Luminex™ 200™ instrument APX10031
Luminex Corporation (sold through Thermo
Fisher Scientific)
MAGPIX™ instrument Luminex Corporation
Use one of the following:
Labnet VorTemp™ 56 Shaking Incubator QP0703 or QP0704 (include Temperature
MaxQ™ 4450 Benchtop Orbital Shaker SHKE4450 or SHKE4450-1CE (required in
Contents and storage

QuantiGene™ Plex Assay kit

The QuantiGene™ Plex Assay Kit is supplied in 3 separate boxes based on storage temperature. Storage conditions are listed below. Refer to the product labels for expiration dating, and refer to the QuantiGene™ Plex Assay Kit Package Insert for individual component volumes or quantities.
Component
Proteinase K
Blocking Reagent Aqueous buered solution containing a preservative -20°C
[1]
Validation Kit)
addition: Universal Platform Cat. No. 30100TS
and Universal Clamps for plates Cat. No. 30175)
Description Storage
Proteinase K in aqueous buered solution -20°C
Label Probe Solution Biotinylated oligonucleotide in aqueous buered
solution
Pre-Amplifier Solution DNA in aqueous buered solution 2-8°C
Amplifier Solution DNA in aqueous buered solution 2-8°C
SAPE Streptavidin-conjugated R-Phycoerythrin 2-8°C
QuantiGene™ Plex Gene Expression Assay User Guide
2-8°C
7
Product information
Contents and storage
(continued)
Component Description Storage
SAPE Diluent Dilution Buer for SAPE reagent 2-8°C
Lysis Mixture Aqueous buered solution containing a preservative 15-30°C
Wash Buer Component 1 Aqueous solution 15-30°C
Wash Buer Component 2 Aqueous buered solution 15-30°C
SAPE Wash Buer Aqueous buered solution 15-30°C
Hybridization Plates 96-well round bottom, clear polypropylene plates 15-30°C
Pressure Seals (Day 1) Clear, pressure-activated seals for use with the
Magnetic Separation Plates 96-well flat bottom microplates 15-30°C
Plate Seals (Day 2) Clear, adhesive plate seals for use with the Magnetic
[1]
We recommend storing in an enzyme storage box, such as the NEB Cool Box (New England Biolabs P/N T0400S). NEVER store at
-80 °C.

QuantiGene™ Plex Panel

The QuantiGene™ Plex Panel includes the target-specific probe set and associated magnetic capture beads. Each panel is supplied in 2 separate boxes based on storage temperature. Refer to the package insert provided with the panel for the gene list and bead identifiers. Do not freeze the capture beads, as they can be damaged if frozen.
Component
Probe set Pre-mixed probe set consisting of target-specific capture
extenders, label extenders, and blocking probes
Capture beads Pre-mixed set of magnetic Luminex™ xMAP™ capture beads
conjugated with capture probes
15-30°C Hybridization Plate during the Day 1/overnight hybridization.
15-30°C Separation Plate during the Day 2 hybridizations
Description Storage
-20°C
2-8°C
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QuantiGene™ Plex Gene Expression Assay User Guide
Before first use
Validate the magnetic plate washer to ensure proper bead retention. For instructions see Appendix B, “Magnetic plate washer validation protocol”.
Calibrate the shaking incubator using the Temperature Validation Kit to ensure hybridization temperatures are 54°C and 50°C. See instructions in the Temperature Validation Kit package insert.
Optimize sample preparation and input by running a dilution series to ensure all targets are within the assay's dynamic range. For sample optimization instructions see “Sample optimization protocol” on page 25.

Sample preparation

Prior to running the QuantiGene™ Plex Assay, ensure you have a lysate or homogenate prepared using one of the following sample processing kits (of note, size/plate refers to 96-well plate format):

Before you begin

Catalog No.
QS0101 Cell Lysate Sample Preparation Kit
QS0102 Cell Lysate Sample Preparation Kit 10 plate
QS0103 Cell Lysate Sample Preparation Kit 5 × 10 plate
QS0104 Fresh or Frozen Tissue Sample Processing Kit
QS0105 Fresh or Frozen Tissue Sample Processing Kit 25 samples
QS0106 Fresh or Frozen Tissue Sample Processing Kit 100 samples
QS0110 Blood Sample Processing Kit
QS0111 Blood Sample Processing Kit 5 plates
QS0112 Blood Sample Processing Kit 5 × 10 Plate
QS0107 FFPE Sample Processing Kit
QS0108 FFPE Sample Processing Kit 25 samples
QS0109 FFPE Sample Processing Kit 100 samples
[1]
Sufficient for preparing bulk lysates from 1.8 x 107 cells or 2 × 96-well plates containing up to 6 × 104 cells/well.
[2]
A sample is defined as 5 mg animal tissue or 15 mg plant tissue.
[3]
A 2-plate kit is sufficient for preparing bulk lysates from 1.8 × 107cells or 2 × 96-well plates containing up to 6 × 104cells/well.
[4]
A sample is defined as 25-100 mm2 × 50-60 microns (area × total thickness of FFPE tissue sections)
Assay specific reagents Size
[1]
[2]
[3]
[4]
2 plate
10 samples
2 plates
10 samples
QuantiGene
Plex Gene Expression Assay User Guide
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Assay procedure: day 1

IMPORTANT!
Tissue homogenates, cell lysates and whole blood lysates must be prepared using the applicable
·
QuantiGene™ Sample Processing Kit. Purified RNA samples do not require a QuantiGene™ Sample Processing Kit.
·
The day 2 procedure is the same for all sample types.
·

For cell lysates or whole blood lysates

1.
Pre-warm Lysis Mixture at 37°C for 30 minutes followed by gentle swirling.
2.
If lysates have been frozen, remove from the freezer and thaw at room temperature followed by incubation at 37°C for 30 min. Following incubation, vortex briefly if samples are in tubes or pipette up and down 5 times if samples are in plates. Leave at room temperature until use.
Do not store on ice prior to use.
3.
Handle the reagents listed below as follows:
a.
Probe Set & Blocking Reagent: Thaw and vortex briefly to mix, then centrifuge Probe Set briefly to collect contents at the bottom of the tube.
b.
Proteinase K: Keep on ice.
c.
Capture Beads: Take out of storage right before use and protect from light.
4.
If samples require dilution, dilute with Diluted Lysis Mixture (dilute 1 volume Lysis Mixture plus 2 volumes Nuclease-free Water, prepared fresh) so that the desired amount of sample is present in a volume of 80 μL/assay well. In order to optimize sample input, please see “Optimize sample input”
on page 25.
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QuantiGene™ Plex Gene Expression Assay User Guide
Assay procedure: day 1
For cell lysates or whole blood lysates
5.
Prepare an appropriate volume of Working Bead Mix by combining the following reagents in the order listed. Scale according to the number of assays to be run, and include sucient overage. Keep Working Bead Mix at room temperature and protected from light. Do not store on ice.
Order Reagent
1 Nuclease-free Water 5.2 624 4. 2 504
2 Lysis Mixture 6.6 792 6.6 792
3 Blocking Reagent 2 240 2 240
4 Proteinase K 0.2 24 0.2 24
5 Capture Beads
(vortex 30 seconds before adding)
6 Probe Set 5 600 6 720
Total 20 2,400 20 2,400
[1]
Includes 25% overage to enable use of reagent reservoir and multichannel pipette.
6.
Vortex Working Bead Mix for 10 seconds, then pipette 20 μL into each well of the Hybridization
1 Well (µL) 96 Well (µL)
2 to 64-plex 65 to 80-plex
[1]
1 120 1 120
1 Well (µL) 96 Well (µL)
Plate.
For fewer than 48 wells: Dispense 20 μL of Working Bead Mix into each well of the Hybridization Plate using a single channel pipette.
For 48 or more wells: Transfer Working Bead Mix to a 25-mL reagent reservoir using a single channel pipette. Do not pour or reagent shortage will occur. Using a multichannel pipette and new tips for each transfer, dispense 20 μL Working Bead Mix into each well of the Hybridization Plate.
[1]
7.
Add 80 μL of lysate or diluted lysate to each well of the Hybridization Plate containing Working Bead Mix. The total final volume in each well will be 100 μL. Load each sample using a new pipette tip.
Background Controls: Add 80μL of Diluted Lysis Mixture (1 volume Lysis Mixture plus 2 volumes Nuclease-free Water) to at least 3 wells containing Working Bead Mix.
8.
Seal the Hybridization Plate using a Pressure Seal: Remove the backing of the Pressure Seal, center and place onto the Hybridization Plate. Using a soft- rubber roller, apply firm even pressure across the seal. Ensure that the plate has been completely sealed.
Note: DO NOT use the Day 2 Plate Seal, otherwise evaporation may occur.
9.
Place the Hybridization Plate in the shaking incubator and incubate for 18-22 hours at 54°C ± 1°C at 600 rpm. Ensure the incubator has been calibrated using the Temperature Validation Kit. If using a VorTemp™ 56, ensure there is an inverted plate lid in place, as explained by the package insert.
10.
After incubation, proceed to “Assay procedure: day 2” on page 16.
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